EP1910570B1 - Gene and polypeptide relating to breast cancer - Google Patents

Gene and polypeptide relating to breast cancer Download PDF

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EP1910570B1
EP1910570B1 EP06782210A EP06782210A EP1910570B1 EP 1910570 B1 EP1910570 B1 EP 1910570B1 EP 06782210 A EP06782210 A EP 06782210A EP 06782210 A EP06782210 A EP 06782210A EP 1910570 B1 EP1910570 B1 EP 1910570B1
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polypeptide
seq
amino acid
acid sequence
polynucleotide
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EP1910570A2 (en
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Yusuke Nakamura
Toyomasa Katagiri
Shuichi Nakatsuru
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Oncotherapy Science Inc
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Oncotherapy Science Inc
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the field of biological science, more specifically to the field of cancer therapy and diagnosis.
  • the present invention relates to novel polypeptides encoded by a novel gene B7330N relating to breast cancer.
  • the invention as described herein relates to the novel gene B7330N.
  • the genes and polypeptides described herein can be used, for example, in the diagnosis of breast cancer, as target molecules for developing drugs against the disease, and for attenuating cell growth of breast cancer.
  • WO 02/059377 A2 and WO 2004/078035 A2 disclose GALNT6 (corresponding to B7330N) as one of several identified genes, which is over-expressed in breast cancer.
  • EP-A-1 621 632 corresponding to WO 2006/013474 shows line up-regulation of GALNT6 (GALNAc) in breast cancer and breast tumour in comparison to normal PBMN cells.
  • WO 2004/094636 discloses siRNAs directed against the GALNT6.
  • Marcos Journal of Histochemistry and Cytochemistry, Histochemical Society, New York; 51; 761-771 (2004 )
  • Bennett JBC; 274; 25362-25370 (1999 ) disclose antibodies and primers which are directed against the polypeptide GALNT6.
  • B7330N was significantly overexpressed in breast cancer cells through the expression profile of breast cancer, and further confirmed that B7330N was overexpressed in breast cancer cells by semi-quantitative RT-PCR and Northern blot analyses.
  • siRNAs effectively inhibited expression of B7330N and suppressed cell/tumor growth of breast cancer.
  • B7330N also designated as GALNT6, as a prominent novel molecular candidate for diagnostic markers and breast cancer drug development.
  • FTIs farnesyltransferase
  • trastuzumab Clinical trials on human using a combination or anti-cancer drugs and anti-HER2 monoclonal antibody, trastuzumab, have been conducted to antagonize the proto-oncogene receptor HER2/neu; and have been achieving improved clinical response and overall survival of breast cancer patients ( Molina MA, et al., Cancer Res 61:4744-4749 (2001 )).
  • a tyrosine kinase inhibitor, ST1-571 which selectively inactivates bcr-abl fusion proteins, has been developed to treat chronic myelogenous leukemias wherein constitutive activation of ber-abl tyrosine kinase plays a crucial role in the transformation of leukocytes.
  • Agents of these kinds are designed to suppress oncogenic activity of specific gene products ( O'Dwyer ME and Druker BJ, Curr Opin Oncol 12:594-7 (2000 )). Therefore, gene products commonly up-regulated in cancerous cells may serve as potential targets for developing novel anti-cancer agents.
  • CTLs cytotoxic T lymphocytes
  • TAAs tumor-associated antigens
  • TAAs are now in the stage of clinical development as targets of immunotherapy. TAAs discovered so far include MAGE ( van der Bruggen et al., Science 254: 1643-7 (1991 )), gp100 ( Kawakami et al., J Exp Med 180: 347-52 (1994 )), SART ( Shichijo et al., J Exp Med 187: 277-88 (1998 )), and NY-ESO-1 ( Chen et al., Proc Natl Acad Sci USA 94: 1914-8 (1997 )). On the other hand, gene products which had been demonstrated to be specifically over-expressed in tumor cells, have been shown to be recognized as targets inducing cellular immune responses.
  • Such gene products include p53 ( Umano et al., Brit J Cancer 84: 1052-7 (2001 )), HER2/neu ( Tanaka et al., Brit J Cancer 84: 94-9 (2001 )), CEA ( Nukaya et al., Int J Cancer 80: 92-7 (1999 )), and so on.
  • TAAs In spite of significant progress in basic and clinical research concerning TAAs ( Rosenberg et al., Nature Med 4: 321-7 (1998 ); Mukherji et al., Proc Natl Acad Sci USA 92: 8078-82 (1995 ); Hu et al., Cancer Res 56: 2479-83 (1996 )), only limited number of candidate TAAs for the treatment of adenocarcinomas, including colorectal cancer, are available. TAAs abundantly expressed in cancer cells, and at the same time which expression is restricted to cancer cells would be promising candidates as immunotherapeutic targets.
  • PBMCs peripheral blood mononuclear cells
  • HLA-A24 and HLA-A0201 are one of the common HLA alleles in Japanese, as well as Caucasian populations ( Date et al., Tissue Antigens 47: 93-101 (1996 ); Kondo et al., J Immunol 155: 4307-12 (1995 ); Kubo et al., J Immunol 152: 3913-24 (1994 ); Imanishi et al., Proceeding of the eleventh International Histocompatibility Workshop and Conference Oxford University Press, Oxford, 1065 (1992 ); Williams et al., Tissue Antigen 49: 129 (1997 )).
  • antigenic peptides of cancers presented by these HLAs may be especially useful for the treatment of cancers among Japanese and Caucasian populations.
  • the induction of low-affinity CTL in vitro usually results from the use of peptide at a high concentration, generating a high level of specific peptide/MHC complexes on antigen presenting cells (APCs), which will effectively activate these CTL (Alexander- Miller et al., Proc Natl Acad Sci USA 93: 4102-7 (1996 )).
  • B7330N also designated as UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 6 ( GALNT6 ) , that was more than three-fold overexpressed in 27 of 77 (35%) breast cancer cases from which we were able to obtain expression data.
  • RNAs small interfering RNAs
  • B7330N encodes a 622-amino acid protein. According to a Northern blot analysis, the expression of B7330N was shown to be restricted to breast cancer cell lines and normal human placenta, pancreas, stomach, trachea, mammary gland and bone marrow.
  • B7330N Many anticancer drugs are not only toxic to cancer cells but also for normally growing cells. However, agents suppressing the expression of B7330N may not adversely affect other organs due to the fact that normal expression of B7330N is restricted to placenta, pancreas, stomach, trachea, mammary gland and bone marrow, and thus may be conveniently used for treating or preventing breast cancer.
  • B7330N which serves as a candidate of diagnostic markers for breast cancer as well as promising potential targets for developing new strategies for diagnosis and effective anti-cancer agents is described herein.
  • a polypeptide encoded by this gene is described herein, as well as the production and the use of the same. More specifically, the novel human polypeptide, B7330N or a functional equivalent thereof is described herein, which expressions are elevated in breast cancer cells.
  • the B7330N polypeptide includes a 622 amino acid protein encoded by the open reading frame of SEQ ID NO: 24 or 26.
  • the B7330N polypeptide preferably includes the amino acid sequence set forth in SEQ ID NO: 25.
  • the present application also provides an isolated protein encoded from at least a portion of the B7330N polynucleotide sequence, or polynucleotide sequences at least 15% and more preferably at least 25% complementary to the sequence set forth in SEQ ID NO: 24 or 26.
  • the present invention further provides a novel human gene B7330N whose expressions is markedly elevated in a great majority of breast cancers as compared to corresponding non-cancerous breast duct epithelium.
  • the isolated B7330N gene includes a polynucleotide sequence as described in SEQ ID NO: 24 or 26.
  • the B7330N cDNA includes 4381 or 4556 nucleotides that contain an open reading frame of 1869 nucleotides (SEQ ID NO: 24 or 26).
  • the invention as described herein further encompasses polynucleotides which hybridize to and which are at least 15% and more preferably at least 25% complementary to the polynucleotide sequence set forth in SEQ ID NO: 24 or 26, to the extent that they encode a B7330N protein or a functional equivalent thereof.
  • polynucleotides are degenerates and allelic mutants of B7330N encoded by the sequence of SEQ ID NO: 24 or 26.
  • an isolated gene is a polynucleotide the structure of which is not identical to that of any naturally occurring polynucleotide or to that of any fragment of a naturally occurring genomic polynucleotide spanning more than three separate genes.
  • the term therefore includes, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule in the genome of the organism in which it naturally occurs; (b) a polynucleotide incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e ., a gene encoding a fusion polypeptide.
  • an isolated polynucleotide that encodes a polypeptide is described herein or a fragment thereof.
  • the isolated polynucleotide includes a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO: 24 or 26. More preferably, the isolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, identical to the nucleotide sequence shown in SEQ ID NO: 24 or 26. In the case of an isolated polynucleotide which is longer than or equivalent in length to the reference sequence, e.g.
  • the comparison is made with the full length of the reference sequence.
  • the comparison is made to a segment of the reference sequence of the same length (excluding any loop required by the homology calculation).
  • a method of producing a protein by transfecting or transforming a host cell with a polynucleotide sequence encoding the B7330N protein is also described herein, and expressing the polynucleotide sequence.
  • vectors comprising a nucleotide sequence encoding the B7330N protein, and host cells harboring a polynucleotides encoding the B7330N protein are also described herein. Such vectors and host cells may be used for producing the B7330N protein.
  • a binding agent that specifically recognizes the B7330N protein is also described herein.
  • a binding agent may be an antibody raised against a B7330N protein.
  • a binding agent may be a ligand specific for the protein, or a synthetic polypeptide that specifically binds the protein ( see e.g., WO2004044011 ).
  • An antisense polynucleotide e.g ., antisense DNA
  • ribozyme ribozyme
  • siRNA small interfering RNA
  • the present invention further provides a method for diagnosis of breast cancer which includes the step of determining an expression level of the gene in a biological sample from a subject, comparing the expression level of B7330N gene with that in a normal sample, and defining that a high expression level of the B7330N gene in the sample indicates that the subject suffers from or is at risk of developing breast cancer.
  • the method includes contacting the B7330N polypeptide with test compounds, and selecting test compounds that bind to or that inhibit the biological activity of the B7330N polypeptide.
  • a method of screening for a compound for use in treating or preventing breast cancer includes contacting a test compound with a cell expressing the B7330N polypeptide or introduced with a vector comprising the transcriptional regulatory region of B7330N upstream of a reporter gene, and selecting the test compound that suppresses the expression level or activity of the reporter gene.
  • a method of screening for a compound for use in treating or preventing breast cancer is also described herein, wherein the method includes contacting a test compound with a B7330N polypeptide, or cell expressing the B7330N polypeptide, and selecting the test compound that suppresses the glycosylation level of the B7330N polypeptide.
  • the glycosylation level is that of asparagine 476 of B7330N polypeptide.
  • a pharmaceutical composition for use in treating or preventing breast cancer is also described herein.
  • the pharmaceutical composition may be, for example, an anti-cancer agent.
  • the pharmaceutical composition can comprise at least a portion of antisense S-oligonucleotides, siRNA molecules or ribozymes against the B7330N polynucleotide sequence shown and described in SEQ ID NO: 24 or 26, respectively.
  • a suitable siRNA targets are a sequence of SEQ ID NOs: 18 or 22.
  • an siRNA as described herein comprises a nucleotide sequence from SEQ ID NOs: 18 or 22. This may be preferably selected as targets for treating or preventing breast cancer according to the invention as described herein.
  • the pharmaceutical compositions may be also those comprising the compounds selected by the present methods of screening for compounds for treating or preventing cell proliferative diseases such as breast cancer.
  • the course of action of the pharmaceutical composition is desirably to inhibit growth of the cancerous cells such as breast cancer cells.
  • the pharmaceutical composition may be applied to mammals including humans and domesticated mammals.
  • the present inventors analyzed the expression profiles of genes in breast cancer using a genome-wide cDNA microarray combined with a laser microbeam microdissection. As a result, B7330N specifically over-expressed in breast cancer cells was identified. Furthermore, suppression of the expression of B7330N gene with small interfering RNAs (siRNAs) resulted in a significant growth-inhibition of cancerous cells.
  • siRNAs small interfering RNAs
  • the B7330N gene is described herein.
  • the cDNA of B7330N consists of 4381 or 4556 nucleotides containing an open reading frame of 1869 nucleotides (SEQ ID NO: 24 or 26; GenBankAccession No. AB265820). These open reading frames encode a 622 amino acid-protein.
  • substantially pure polypeptides encoded by these genes including polypeptides comprising the amino acid sequence of SEQ ID NO: 25, as well as functional equivalent thereof, to the extend that they encode a B7330N protein are described herein.
  • polypeptides functionally equivalent to B7330N include, for example, homologous proteins of other organisms corresponding to the human B7330N protein, as well as mutants of human B7330N proteins.
  • the term "functionally equivalent" as described herein means that the subject polypeptide has the activity to promote cell proliferation like the B7330N protein and to confer oncogenic activity to cancer cells. Whether the subject polypeptide has a cell proliferation activity or hot can be judged by introducing the DNA encoding the subject polypeptide into a cell, expressing the respective polypeptide and detecting promotion of proliferation of the cells or increase in colony forming activity.
  • Such cells include, for example, NIH3T3, COS7 and HEK293.
  • polypeptides functionally equivalent to a given protein are well known by a person skilled in the art and include known methods of introducing mutations into the protein.
  • one skilled in the art can prepare polypeptides functionally equivalent to the human B7330N protein by introducing an appropriate mutation in the amino acid sequence of these proteins by site-directed mutagenesis ( Hashimoto-Gotoh et al., Gene 152:271-5 (1995 ); Zoller and Smith, Methods Enzymol 100: 468-500 (1983 ); Kramer et al., Nucleic Acids Res.
  • the polypeptide described herein includes those proteins having the amino acid sequences of the human B7330N protein in which one or more amino acids are mutated, provided the resulting mutated polypeptides are functionally equivalent to the human B7330N protein.
  • the number of amino acids to be mutated in such a mutant is generally 10 amino acids or less, preferably 6 amino acids or less, and more preferably 3 amino acids or less.
  • Mutated or modified proteins proteins having amino acid sequences modified by substituting, deleting, inserting and/or adding one or more amino acid residues of a certain amino acid sequence, have been known to retain the original biological activity ( Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984 ); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982 ); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982 )).
  • the amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution).
  • properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W).
  • the parenthetic letters indicate the one-letter codes of amino acids.
  • a preferable functional equivalent of the B7330N protein as described herein conserves a glycosylation site thereof. For example, it was confirmed that B7330N protein was glycosylated at 476N. Accordingly, in the preferable embodiments, a functional equivalent of B7330N protein consists of an amino acid sequence comprising 476N or homologous to the 476N in a homologous sequence. The amino acid sequence of the functional equivalent of a B7330N protein, a position homologous to the 476 th position can be determined by comparing the amino acid sequences. The position in a protein of interest needs not necessarily be the 476 th position.
  • the homologous position may be a position other than the 476 th position.
  • amino acid sequences of both proteins are aligned so as to match mutual amino acids as well as amino acids having similar properties as much as possible by inserting appropriate gaps in both amino acid sequences if necessary.
  • it can be determined which position in a protein of interest corresponds to a position homologous to the 476 th position in the B7330N protein.
  • Such a technique has been known among those skilled in the art, and can be performed easily using commercially available or published computer software, for example, the analytical software GENETYX-MAC VER. 10 (Software), etc.
  • polypeptide to which one or more amino acids residues are added to the amino acid sequence of human B7330N protein is a fusion protein containing the human B7330N protein.
  • Fusion proteins, fusions of the human B7330N protein and other peptides or proteins, are included in the present invention. Fusion proteins can be made by techniques well known to a person skilled in the art, such as by linking the DNA encoding the human B7330N protein of the invention with DNA encoding other peptides or proteins, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins fused to the protein of the present invention.
  • peptides that can be used as peptides that are fused to the protein of the present invention include, for example, FLAG ( Hopp et al., Biotechnology 6: 1204-10 (1988 )), 6xHis containing six His (histidine) residues, 10xHis, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, ⁇ -tubulin fragment, B-tag, Protein C fragment and the like.
  • FLAG Hopp et al., Biotechnology 6: 1204-10 (1988 )
  • 6xHis containing six His (histidine) residues 10xHis
  • Influenza agglutinin (HA) Influenza agglutinin
  • human c-myc fragment VSP-GP fragment
  • p18HIV fragment T7-tag
  • HSV-tag HSV-tag
  • Fusion proteins can be prepared by fusing commercially available DNA, encoding the fusion peptides or proteins discussed above, with the DNA encoding the polypeptide of the present invention and expressing the fused DNA prepared.
  • polypeptides described herein include those that are encoded by DNA that hybridizes with a whole or part of the DNA sequence encoding the human B7330N protein and are functionally equivalent to the human B7330N protein.
  • These polypeptides include mammal homologues corresponding to the protein derived from human (for example, a polypeptide encoded by a monkey, rat, rabbit and bovine gene).
  • mammal homologues corresponding to the protein derived from human (for example, a polypeptide encoded by a monkey, rat, rabbit and bovine gene).
  • isolating a cDNA highly homologous to the DNA encoding the human B7330N protein from animals it is particularly preferable to use tissues from breast cancer cell and normal human placenta, pancreas, stomach, trachea, mammary gland and bone marrow.
  • hybridization for isolating a DNA encoding a polypeptide functionally equivalent to the human B7330N protein can be routinely selected by a person skilled in the art.
  • hybridization may be performed by conducting prehybridization at 68°C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68°C for 1 hour or longer.
  • the following washing step can be conducted, for example, in a low stringent condition.
  • a low stringent condition is, for example, 42°C, 2 x SSC, 0.1% SDS, or preferably 50°C, 2 x SSC, 0.1% SDS. More preferably, high stringent conditions are used.
  • a high stringent condition is, for example, washing 3 times in 2 x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37°C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50°C for 20 min.
  • factors such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
  • a gene amplification method for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a DNA encoding a polypeptide functionally equivalent to the human B7330N protein, using a primer synthesized based on the sequence information of the protein encoding DNA (SEQ ID NO: 24 or 26).
  • PCR polymerase chain reaction
  • Polypeptides that are functionally equivalent to the human B7330N protein encoded by the DNA isolated through the above hybridization techniques or gene amplification techniques normally have a high homology to the amino acid sequence of the human B7330N protein.
  • "High homology” typically refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 85%, 90%, 93%, 95%, 98%, 99% or higher between a polypeptide sequence or a polynucleotide sequence and a reference sequence.
  • Percent homology also referred to as percent identity
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences and comparison can be conducted, e.g ., using the algorithm in " Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983 )".
  • a polypeptide as described herein has variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has a function equivalent to that of the human B7330N protein of described herein, it is within the scope of the invention.
  • the polypeptide described herein can be prepared as recombinant protein or natural protein, by methods well known to those skilled in the art.
  • a recombinant protein can be prepared by inserting a DNA, which encodes the polypeptide described herein (for example, the DNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26), into an appropriate expressions vector, introducing the vector into an appropriate host cell, obtaining the extract, and purifying the polypeptide by subjecting the extract to chromatography, e.g ., ion exchange chromatography, reverse phase chromatography, gel filtration or affinity chromatography utilizing a column to which antibodies against the protein described herein is fixed or by combining more than one of aforementioned columns.
  • chromatography e.g ., ion exchange chromatography, reverse phase chromatography, gel filtration or affinity chromatography utilizing a column to which antibodies against the protein described herein is fixed or by combining more than one of aforementioned columns.
  • polypeptide described herein when expressed within host cells (for example, animal cells and E. coli ) as a fusion protein with glutathione-S-transferase protein or as a recombinant protein supplemented with multiple histidines, the expressed recombinant protein can be purified using a glutathione column or nickel column.
  • host cells for example, animal cells and E. coli
  • the polypeptide described herein when expressed as a protein tagged with c-myc, multiple histidines or FLAG, it can be detected and purified using antibodies to c-myc, His or FLAG, respectively.
  • a natural protein can be isolated by methods known to a person skilled in the art, for example, by contacting the affinity column, in which antibodies binding to the B7330N protein described below are bound, with the extract of tissues or cells expressing the polypeptide of the present invention.
  • the antibodies can be polyclonal antibodies or monoclonal antibodies.
  • Partial peptides of the polypeptide described herein are also encompassed.
  • the partial peptide has an amino acid sequence specific to the polypeptide of the present invention and consists of at least 7 amino acids, preferably 8 amino acids or more, and more preferably 9 amino acids or more.
  • the partial peptide can be used, for example, for preparing antibodies against the polypeptide described herein screening for a compound that binds to the polypeptide described herein, and screening for inhibitors of the polypeptide described herein.
  • a partial peptide as described herein can be produced by genetic engineering, by known methods of peptide synthesis or by digesting the polypeptide of the invention with an appropriate peptidase.
  • peptide synthesis for example, solid phase synthesis or liquid phase synthesis may be used.
  • polynucleotides that encode such B7330N polypeptides described above are also described herein.
  • the polynucleotides described herein can be used for the in vivo or in vitro production of the polypeptide as described above, or can be applied to gene therapy for diseases attributed to genetic abnormality in the gene encoding the protein described herein. Any form of the polynucleotide described herein can be used so long as it encodes the polypeptide described herein, including mRNA, RNA, cDNA, genomic DNA, chemically synthesized polynucleotides.
  • the polynucleotide described herein includes a DNA comprising a given nucleotide sequences as well as its degenerate sequences, so long as the resulting DNA encodes a polypeptide as described herein.
  • the polynucleotide described herein can be prepared by methods known to a person skilled in the art
  • the polynucleotide described herein can be prepared by: preparing a cDNA library from cells which express the polypeptide of the present invention, and conducting hybridization using a partial sequence of the DNA of the present invention (for example, SEQ ID NO: 24 or 26) as a probe.
  • a cDNA library can be prepared, for example, by the method described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989 ); alternatively, commercially available cDNA libraries may be used.
  • a cDNA library can be also prepared by: extracting RNAs from cells expressing the polypeptide of the present invention, synthesizing oligo DNAs based on the sequence of the DNA described herein (for example, SEQ ID NO: 24 or 26), conducting PCR using the oligo DNAs as primers, and amplifying cDNAs encoding the protein of the present invention.
  • the translation region encoded by the cDNA can be routinely determined, and the amino acid sequence of the polypeptide described herein can be easily obtained.
  • the genomic DNA can be isolated.
  • mRNAs may first be prepared from a cell, tissue or organ (e.g ., breast cancer cell and normal human placenta, pancreas, stomach, trachea, mammary gland and bone marrow) in which the object polypeptide of the invention is expressed.
  • a cell, tissue or organ e.g ., breast cancer cell and normal human placenta, pancreas, stomach, trachea, mammary gland and bone marrow
  • Known methods can be used to isolate mRNAs; for instance, total RNA may be prepared by guanidine ultracentrifugation ( Chirgwin et al., Biochemistry 18:5294-9 (1979 )) or AGPC method ( Chomczynski and Sacchi, Anal Biochem 162:156-9 (1987 )).
  • mRNA may be purified from total RNA using mRNA Purification Kit (Pharmacia) and such.
  • mRNA may be directly purified by QuickPrep mRNA Purification Kit (P
  • cDNA is used to synthesize cDNA using reverse transcriptase.
  • cDNA may be synthesized using a commercially available kit, such as the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).
  • cDNA may be synthesized and amplified following the 5'-RACE method ( Frohman et al., Proc Natl Acad Sci USA 85: 8998-9002 (1988 ); Belyavsky et al., Nucleic Acids Res. 17: 2919-32 (1989 )), which uses a primer and such, described herein, the 5'-Ampli FINDER RACE Kit (Clontech), and polymerase chain reaction (PCR).
  • 5'-RACE method Frohman et al., Proc Natl Acad Sci USA 85: 8998-9002 (1988 ); Belyavsky et al., Nucleic Acids Res. 17: 2919-32
  • a desired DNA fragment is prepared from the P CR products and ligated with a vector DNA.
  • the recombinant vectors are used to transform E. coli and such, and a desired recombinant vector is prepared from a selected colony.
  • the nucleotide sequence of the desired DNA can be verified by conventional methods, such as dideoxynucleotide chain termination.
  • the nucleotide sequence of a polynucleotide described herein may be designed to be expressed more efficiently by taking into account the frequency of codon usage in the host to be used for expression ( Grantham et al., Nucleic Acids Res. 9: 43-74 (1981 )).
  • the sequence of the polynucleotide described herein may be altered by a commercially available kit or a conventional method. For instance, the sequence may be altered by digestion with restriction enzymes, insertion of a synthetic oligonucleotide or an appropriate polynucleotide fragment, addition of a linker, or insertion of the initiation codon (ATG) and/or the stop codon (TAA, TGA or TAG).
  • polynucleotide described herein encompasses the DNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26.
  • a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a nucleotide sequence of SEQ ID NO: 24 or 26, and encodes a polypeptide functionally equivalent to the B7330N protein described above is also described herein.
  • stringent conditions For example, low stringent condition can be used. More preferably, high stringent condition can be used. These conditions are the same as that described above.
  • the hybridizing DNA above is preferably a cDNA or a chromosomal DNA.
  • a polynucleotide which is complementary to the polynucleotide encoding human B7330N protein (SEQ ID NO: 24 or 26) or the complementary strand thereof, and which comprises at least 15 nucleotides is also described herein.
  • the polynucleotide as described herein is preferably a polynucleotide which specifically hybridizes with the DNA encoding the B7330N polypeptide described herein.
  • the term “specifically hybridize” as used herein, means that cross-hybridization does not occur significantly with DNA encoding other proteins, under the usual hybridizing conditions, preferably under stringent hybridizing conditions.
  • Such polynucleotides include, probes, primers, nucleotides and nucleotide derivatives (for example, antisense oligonucleotides and ribozymes), which specifically hybridize with DNA encoding the polypeptide described herein or its complementary strand. Moreover, such polynucleotide can be utilized for the preparation of DNA chip.
  • a vector and host cell into which a polynucleotide of the invention is introduced are also described herein.
  • a vector as described herein is useful to keep a polynucleotide, especially a DNA, as described herein in host cell, to express the polypeptide described herein, or to administer the polynucleotide described herein for gene therapy.
  • E. coli When E. coli is a host cell and the vector is amplified and produced in a large amount in E. coli (e.g., JM109, DH5 ⁇ , HB101 or XL1Blue), the vector should have "ori" to be amplified in E. coli and a marker gene for selecting transformed E. coli (e.g., a drug-resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol or the like).
  • a marker gene for selecting transformed E. coli e.g., a drug-resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol or the like.
  • M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, etc. can be used.
  • pGEM-T pDIRECT and pT7 can also be used for subcloning and extracting cDNA as well as the vectors described above.
  • an expression vector is especially useful.
  • an expression vector to be expressed in E. coli should have the above characteristics to be amplified in E. coli.
  • the vector should have a promoter, for example, lacZ promoter ( Ward et al., Nature 341: 544-6 (1989 ); FASEB J 6: 2422-7 (1992 )), araB promoter ( Better et al., Science 240: 1041-3 (1988 )), T7 promoter or the like, that can efficiently express the desired gene in E. coli.
  • a promoter for example, lacZ promoter ( Ward et al., Nature 341: 544-6 (1989 ); FASEB J 6: 2422-7 (1992 )), araB promoter ( Better et al., Science 240: 1041-3 (1988 )), T7 promoter or the like, that can efficiently express the desired gene in E. coli.
  • the host is preferably BL21 which expresses T7 RNA polymerase
  • the vector may also contain a signal sequence for polypeptide secretion.
  • An exemplary signal sequence that directs the polypeptide to be secreted to the periplasm of the E. coli is the pelB signal sequence ( Lei et al., J Bacteriol. 169: 4379 (1987 )).
  • Means for introducing of the vectors into the target host cells include, for example, the calcium chloride method, and the electroporation method.
  • expression vectors derived from mammals for example, pcDNA3 (Invitrogen) and pEF-BOS ( Mizushinna S and Nagata S., Nucleic Acids Res 18(17): 5322 (1990 )
  • pEF, pCDM8 expression vectors derived from insect cells
  • expression vectors derived from plants for example, "Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8)
  • expression vectors derived from plants e.g ., pMH1, pMH2
  • expression vectors derived from animal viruses e.g ., pHSV, pMV, pAdexLcw
  • expression vectors derived from retroviruses e.g ., pZIpneo
  • expression vector derived from yeast e.g ., "Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01) and expression vectors derived from
  • the vector In order to express the vector in animal cells, such as CHO, COS or NIH3T3 cells, the vector should have a promoter necessary for expression in such cells, for example, the SV40 promoter ( Mulligan et al., Nature 277: 108 (1979 )), the MMLV-LTR promoter, the EF1 ⁇ promoter ( Mizushima et al., Nucleic Acids Res 18: 5322 (1990 )), the CMV promoter and the like, and preferably a marker gene for selecting transformants (for example, a drug resistance gene selected by a drug ( e.g ., neomycin, G418)).
  • a promoter necessary for expression in such cells for example, the SV40 promoter ( Mulligan et al., Nature 277: 108 (1979 )), the MMLV-LTR promoter, the EF1 ⁇ promoter ( Mizushima et al., Nucleic Acids Res 18: 5322 (1990 )), the
  • the polypeptides may be prepared by culturing a host cell which harbors an expression vector comprising a gene encoding the polypeptide. According to needs, methods may be used to express a gene stably and, at the same time, to amplify the copy number of the gene in cells.
  • a vector comprising the complementary DHFR gene e.g ., pCHO I
  • MTX methotrexate
  • a vector comprising a replication origin of SV40 pcD, etc.
  • COS cells comprising the SV40 T antigen expressing gene on the chromosome
  • a polypeptides as described herein obtained as above may be isolated from inside or outside (such as medium) of host cells and purified as a substantially pure homogeneous polypeptide.
  • the term "substantially pure” as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules.
  • the substantially pure polypeptide is at least 75% (e.g ., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • the method for polypeptide isolation and purification is not limited to any specific method; in fact, any standard method may be used.
  • column chromatography filter, ultrafiltration, salt precipitation, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization may be appropriately selected and combined to isolate and purify the polypeptide.
  • chromatography examples include, for example, affinity chromatography, ionexchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and such ( Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996 )). These chromatographies may be performed by liquid chromatography, such as HPLC and FPLC. Thus, methods for preparing highly purified polypeptides are described above.
  • a polypeptide described herein may be optionally modified or partially deleted by treating it with an appropriate protein modification enzyme before or after purification.
  • useful protein modification enzymes include, but are not limited to, trypsin, chymotrypsin, lysylendopeptidase, protein kinase, glucosidase and so on.
  • the antibody described herein can be used in any form, such as monoclonal or polyclonal antibodies, and includes antiserum obtained by immunizing an animal such as a rabbit with the polypeptide of the invention, all classes of polyclonal and monoclonal antibodies, human antibodies and humanized antibodies produced by genetic recombination.
  • a polypeptide described hereinused as an antigen to obtain an antibody may be derived from any animal species, but preferably is derived from a mammal such as a human, mouse, or rat, more preferably from a human.
  • a human-derived polypeptide may be obtained from the nucleotide or amino acid sequences disclosed herein.
  • polypeptide described herein to be used as an immunization antigen may be a complete protein or a partial peptide of the protein.
  • a partial peptide may comprise, for example, the amino (N)-terminal or carboxy (C)-terminal fragment of a polypeptide described herein.
  • an antibody is defined as a protein that reacts with either the full length or a fragment of a polypeptide described herein.
  • a gene encoding a polypeptidedescribed hereinor its fragment may be inserted into a known expression vector, which is then used to transform a host cell as described herein.
  • the desired polypeptide or its fragment may be recovered from the outside or inside of host cells by any standard method, and may subsequently be used as an antigen.
  • whole cells expressing the polypeptide or their lysates or a chemically synthesized polypeptide may be used as the antigen.
  • Any mammalian animal may be immunized with the antigen, but preferably the compatibility with parental cells used for cell fusion is taken into account.
  • animals of Rodentia, Lagomorpha or Primates are used.
  • Animals of Rodentia include, for example, mouse, rat and hamster.
  • Animals of Lagomorpha include, for example, rabbit
  • Animals of Primates include, for example, a monkey of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkey, sacred baboon and chimpanzees.
  • antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc.
  • PBS phosphate buffered saline
  • the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion and then administered to mammalian animals.
  • a standard adjuvant such as Freund's complete adjuvant
  • an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days.
  • An appropriate carrier may also be used for immunization.
  • serum is examined by a standard method for an increase in the amount of desired antibodies.
  • Polyclonal antibodies against the polypeptides described herein may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method.
  • Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies may be isolated from the serum.
  • Immunoglobulin G or M can be prepared from a fraction which recognizes only the polypeptide described herein using, for example, an affinity column coupled with the polypeptide of the present invention, and further purifying this fraction using protein A or protein G column.
  • immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion.
  • the immune cells used for cell fusion are preferably obtained from spleen.
  • Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.
  • the above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al. ( Galfre and Milstein, Methods Enzymol 73: 3-46 (1981 )).
  • Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin and thymidine containing medium).
  • HAT medium hyperxanthine, aminopterin and thymidine containing medium.
  • the cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma (non-fused cells), to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.
  • human lymphocytes such as those infected by EB virus may be immunized with a polypeptide, polypeptide expressing cells or their lysates in vitro . Then, the immunized lymphocytes are fused with human-derived myeloma cells that are capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody that is able to bind to the polypeptide can be obtained (Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688 ).
  • the obtained hybridomas are subsequently transplanted into the abdominal cavity of a mouse and the ascites are extracted.
  • the obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography or an affinity column to which the polypeptide of the present invention is coupled.
  • the antibody, described herein can be used not only for purification and detection of the polypeptide described herein, but also as a candidate for agonists and antagonists of the polypeptide, described herein.
  • this antibody can be applied to the antibody treatment for diseases related to the polypeptide described herein.
  • a human antibody or a humanized antibody is preferable for reducing immunogenicity.
  • transgenic animals having a repertory of human antibody genes may be immunized with an antigen selected from a polypeptide, polypeptide expressing cells or their lysates.
  • Antibody producing cells are then collected from the animals and fused with myeloma cells to obtain hybridoma, from which human antibodies against the polypeptide can be prepared ( see WO92-03918 , WO94-02602 , WO94-25585 , WO96-33735 and WO96-34096 ).
  • an immune cell such as an immunized lymphocyte, producing antibodies may be immortalized by an oncogene and used for preparing monoclonal antibodies.
  • Monoclonal antibodies thus obtained can be also recombinantly prepared using genetic engineering techniques (see , for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1990 )).
  • a DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody.
  • the present invention also provides recombinant antibodies prepared as described above.
  • an antibody described herein may be a fragment of an antibody or modified antibody, so long as it binds to one or more of the polypeptides of the invention.
  • the antibody fragment may be Fab, F(ab') 2 , Fv or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker ( Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988 )).
  • an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin.
  • a gene encoding the antibody fragment may be constructed, inserted into an expression vector and expressed in an appropriate host cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994 ); Better and Horwitz, Methods Enzymol 178: 476-96 (1989 ); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989 ); Lamoyi, Methods Enzymol 121: 652-63 (1986 ); Rousseaux et al., Methods Enzymol 121: 663-9 (1986 ); Bird and Walker, Trends Biotechnol 9: 132-7 (1991 )).
  • An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). Such modified antibodies are also described herein.
  • PEG polyethylene glycol
  • the modified antibody can be obtained by chemically modifying an antibody. These modification methods are conventional in the field.
  • an antibody as described herein may be obtained as a chimeric antibody, between a variable region derived from nonhuman antibody and the constant region derived from human antibody, or as a humanized antibody, comprising the complementarity determining region (CDR) derived from nonhuman antibody, the frame work region (FR) and the constant region derived from human antibody.
  • CDR complementarity determining region
  • FR frame work region
  • Such antibodies can be prepared according to known technology. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody ( see e.g., Verhoeyen et al., Science 239:1534-6 (1988 )). Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • Fully human antibodies comprising human variable regions in addition to human framework and constant regions can also be used. Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage ( e.g ., Hoogenboom & Winter, J. Mol. Biol. 227:381 (1992 )). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g ., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g ., in U.S. Patent Nos. 6,150,584 , 5,545,807 ; 5,545,806 ; 5,569,825 ; 5,625,126 ; 5,633,425 ; 5,661,016 .
  • Antibodies obtained as above may be purified to homogeneity.
  • the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins.
  • the antibody may be separated and isolated by the appropriately selected and combined use of column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis and isoelectric focusing ( Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988 )), but are not limited thereto.
  • a protein A column and protein G column can be used as the affinity column.
  • Exemplary protein A columns to be used include, for example, Hyper D, POROS and Sepharose F.F. (Pharmacia).
  • Exemplary chromatography with the exception of affinity includes, for example, ionexchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography and the like ( Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996 )).
  • the chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • the antibody, described herein is immobilized on a plate, a polypeptide of the invention is applied to the plate, and then a sample containing a desired antibody, such as culture supernatant of antibody producing cells or purified antibodies, is applied. Then, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme, such as alkaline phosphatase, is applied, and the plate is incubated.
  • a desired antibody such as culture supernatant of antibody producing cells or purified antibodies
  • an enzyme substrate such asp-nitrophenyl phosphate
  • the absorbance is measured to evaluate the antigen binding activity of the sample.
  • a fragment of the polypeptide such as a C-terminal or N-terminal fragment, may be used as the antigen to evaluate the binding activity of the antibody.
  • BIAcore Pharmacia
  • the above methods allow for the detection or measurement of the polypeptide of the invention, by exposing the antibody of the invention to a sample assumed to contain the polypeptide of the invention, and detecting or measuring the immune complex formed by the antibody and the polypeptide.
  • the method of detection or measurement of the polypeptide according to the invention can specifically detect or measure a polypeptide, the method may be useful in a variety of experiments in which the polypeptide is used.
  • an antisense oligonucleotide that hybridizes with any site within the nucleotide sequence of SEQ ID NO: 24 or 26 is also described herein.
  • This antisense oligonucleotide is preferably against at least about 15 continuous nucleotides of the nucleotide sequence of SEQ ID NO: 24 or 26.
  • the above-mentioned antisense oligonucleotide, which contains an initiation codon in the above-mentioned at least 15 continuous nucleotides, is even more preferred.
  • Derivatives or modified products of antisense oligonucleotides can also be used as antisense oligonucleotides.
  • modified products include lower alkyl phosphonate modifications such as methyl-phosphonate-type or ethyl-phosphonate-type, phosphorothioate modifications and phosphoroamidate modifications.
  • antisense oligonucleotides means, not only those in which the nucleotides corresponding to those constituting a specified region of a DNA or mRNA are entirely complementary, but also those having a mismatch of one or more nucleotides, as long as the DNA or mRNA and the antisense oligonucleotide can specifically hybridize with the nucleotide sequence of SEQ ID NO: 24 or 26.
  • Such polynucleotides are contained as those having, in the "at least about 15 continuous nucleotide sequence region", a homology of at least 70% or higher, preferably at 80% or higher, more preferably about 90% or higher, even more preferably about 95% or higher.
  • the algorithm stated herein can be used to determine the homology.
  • Algorithms known in the art can be used to determine the homology.
  • derivatives or modified products of the antisense-oligonucleotides can also be used herein as antisense-oligonucleotides Examples of such modified products include lower alkyl phosphonate modifications such as methyl-phosphonate-type or ethyl-phosphonate-type, phosphorothioate modifications and phosphoroamidate modifications.
  • antisense polynucleotides are useful as probes for the isolation or detection of DNA encoding the polypeptide of the invention or as a primer used for amplifications.
  • the antisense oligonucleotide derivatives of the present invention act upon cells producing the polypeptide of the invention by binding to the DNA or mRNA encoding the polypeptide, inhibiting its transcription or translation, promoting the degradation of the mRNA and inhibiting the expression of the polypeptide of the invention, thereby resulting in the inhibition of the polypeptide's function.
  • siRNA Small interfering RNAs
  • siRNA Small interfering RNAs
  • such siRNA for suppressing the expression of B7330N include those that target the nucleotide sequence of SEQ ID NOs: 18 or 22.
  • siRNAs refers to a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques are used for introducing siRNA into cells, including those wherein DNA is used as the template to transcribed RNA.
  • the siRNA comprises a sense nucleic acid sequence and an antisense nucleic acid sequence of the polynucleotide encoding human B7330N protein (SEQ ID NO: 24 or 26).
  • the siRNA is constructed such that a single transcript (double stranded RNA) has both the sense and complementary antisense sequences from the target gene, e.g ., a hairpin.
  • the length of the oligonucleotide is at least 10 nucleotides and may be as long as the naturally-occurring the transcript. Preferably, the oligonucleotide is less than about 75, about 50, about 25 nucleotides in length. Most preferably, the oligonucleotide is about 19 to about 25 nucleotides in length.
  • B7330N siRNA oligonucleotide which inhibit the growth of the cancer cell include the target sequence containing SEQ ID NOs: 18 or 22.
  • nucleotide "u” can be added to 3'end of the antisense strand of the target sequence.
  • the number of “u”s to be added is at least about 2, generally about 2 to about 10, preferably about 2 to about 5.
  • the added "u”s form single strand at the 3'end of the antisense strand of the siRNA.
  • a B7330N siRNA is directly introduced into the cells in a form that is capable of binding to the mRNA transcripts.
  • the siRNA molecules described herein are typically modified as described above for antisense molecules.
  • Other modifications are also possible, for example, cholesterol-conjugated siRNAs have shown improved pharmacological properties ( Song et al. Nature Med. 9:347-51 (2003 )).
  • the DNA encoding the B7330N siRNA is in a vector.
  • Vectors are produced for example by cloning a B7330N target sequence into an expression vector operatively-linked regulatory sequences flanking the B7330N sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands ( Lee, N.S. et al., (2002) Nature Biotechnology 20 : 500-5 .).
  • RNA molecule that is antisense to B7330N mRNA is transcribed by a first promoter (e.g ., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the B7330N mRNA is transcribed by a second promoter (e.g ., a promoter sequence 5' of the cloned DNA).
  • a first promoter e.g ., a promoter sequence 3' of the cloned DNA
  • RNA molecule that is the sense strand for the B7330N mRNA is transcribed by a second promoter (e.g ., a promoter sequence 5' of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate siRNA constructs for silencing of the B7330N gene.
  • siRNA construct Cloned B7330N can encode a construct having secondary structure, e.g ., hairpins, wherein a single transcript has both the sense and complementary antisense sequences from the target gene.
  • a loop sequence consisting of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure.
  • the present invention also provides siRNA having the general formula 5'-[A]-[B]-[A']-3', wherein [A] is a ribonucleotide sequence corresponding to a sequence that specifically hybridizes to an mRNA or a cDNA from a B7330N gene.
  • [A] is a ribonucleotide sequence corresponding a sequence of nucleotides 417-435 of SEQ ID NO: 24 or 623-641 of SEQ ID NO: 26 (SEQ ID NO: 18) and 1366-1384 of SEQ ID NO: 24 or 1572-1590 of SEQ ID NO: 26 (SEQ ID NO: 22),
  • [B] is a ribonucleotide sequence consisting of about 3 to about 23 nucleotides
  • [A'] is a ribonucleotide sequence consisting of the complementary sequence of [A].
  • the loop sequence may consist of arbitrary sequence having preferably 3 to 23 nucleotide in length.
  • the loop sequence for example, can be selected from group consisting of following sequences (http://www.ambion.com/techlib/tb/tb_506.html).
  • nucleotide "u" can be added to the 3'end of [A'], in order to enhance the inhibiting activity of the siRNA.
  • the number of "u"s to be added is at least about 2, generally about 2 to about 10, preferably about 2 to about 5.
  • loop sequence consisting of 23 nucleotides also provides active siRNA ( Jacque, J. M., et al., (2002) Nature 418: 435-8 .).
  • the loop sequence can be selected from group consisting of CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA.
  • Preferable loop sequence is UUCAAGAGA ("ttcaagaga" in DNA).
  • siRNAs are transcribed intracellularly by cloning the B7330N gene templates into a vector containing, e.g ., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
  • transfection-enhancing agent can be used. FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent.
  • nucleotide sequence of siRNAs may be designed using an siRNA design computer program available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html). Nucleotide sequences for the siRNA are selected by the computer program based on the following protocol:
  • Oligonucleotides and oligonucleotides complementary to various portions of B7330N mRNA were tested in vitro for their ability to decrease production of B7330N in tumor cells (e.g ., using the HBL-100, HCC1937, MCF-7, MDA-MB-435s, YMB1, SKBR3, T47D, BT-20, BT-474, BT-549, HCC1143, HCC1500, HCC1599, MDA-MB-157, MDA-MB453, OUCB-F, ZR-75-1 breast cancer cell line) according to standard methods.
  • a reduction in B7330N gene product in cells contacted with the candidate siRNA composition compared to cells cultured in the absence of the candidate composition is detected using B7330N-specific antibodies or other detection strategies. Sequences which decrease production of B7330N in in vitro cell-based or cell-free assays are then tested for there inhibitory effects on cell growth. Sequences which inhibit cell growth in in vitro cell-based assay are test in in vivo in rats or mice to confirm decreased B7330N production and decreased tumor cell growth in animals with malignant neoplasms.
  • double-stranded molecules that include the nucleic acid sequence of target sequences, for example, nucleotides 417-435 of SEQ ID NO: 24 or 623-641 of SEQ ID NO: 26 (SEQ ID NO: 18) and 1366-1384 of SEQ ID NO: 24 or 1572-1590 of SEQ ID NO: 26 (SEQ ID NO: 22).
  • the double-stranded molecule described herein comprises a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to SEQ ID NOs: 18, or 22 and wherein the antisense strand comprises a ribonucleotide sequence which is complementary to said sense strand, wherein said sense strand and said antisense strand hybridize to each other to form said double-stranded molecule, and wherein said double-stranded molecule, when introduced into a cell expressing the B7330N gene, inhibits expression of said gene.
  • the isolated nucleic acid is RNA or derivatives thereof, base "t" should be replaced with "u” in the nucleotide sequences.
  • the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule
  • binding means the physical or chemical interaction between two nucleic acids or compounds or associated nucleic acids or compounds or combinations thereof.
  • Complementary nucleic acid sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches.
  • the sense strand and antisense strand of the isolated nucleotide described herein can form double stranded nucleotide or hairpin loop structure by the hybridization.
  • such duplexes contain no more than 1 mismatch for every 10 matches.
  • the strands of the duplex are fully complementary, such duplexes contain no mismatches.
  • the nucleic acid molecule is less than 4381 nucleotides (for SEQ ID NO: 24) or 4556 nucleotides (for SEQ ID NO: 26) in length.
  • the nucleic acid molecule is less than 500, 200, or 75 nucleotide in length.
  • a vector containing one or more of the nucleic acids described herein is also included herein, and a cell containing the vectors.
  • the isolated nucleic acids described herein are useful for siRNA against B7330N or DNA encoding the siRNA.
  • the sense strand is preferably longer than about 19 nucleotides, and more preferably longer than about 21 nucleotides.
  • the antisense oligonucleotide or siRNA of the invention inhibit the expression of the polypeptide of the invention and is thereby useful for suppressing the biological activity of the polypeptide of the invention.
  • expression-inhibitors comprising the antisense oligonucleotide or siRNA of the invention, are useful in the point that they can inhibit the biological activity of the polypeptide of the invention. Therefore, a composition comprising antisense oligonucleotide or siRNA of the present invention are useful in treating a breast cancer.
  • Examples of B7330N siRNA oligonucleotide which inhibit the expression in mammalian cells include the target sequence containing SEQ ID NOs: 18 or 22.
  • nucleotide "u” can be added to 3'end of the antisense strand of the target sequence.
  • the number of “u”s to be added is at least about 2, generally about 2 to about 10, preferably about 2 to about 5.
  • the added "u”s form single strand at the 3' end of the antisense strand of the siRNA.
  • expression-inhibitors comprising the antisense oligonucleotide or siRNA of the invention, are useful in the point that they can inhibit the biological activity of the polypeptide of the invention.
  • a composition comprising the antisense oligonucleotide or siRNA. described herein is useful in treating a cell proliferative disease such as breast cancer.
  • ribozymes are described herein that inhibit the expression of the B7330N polypeptide described herein.
  • ribozymes are classified into large ribozymes and small ribozymes.
  • a large ribozyme is known as an enzyme that cleaves the phosphate ester bond of nucleic acids. After the reaction with the large ribozyme, the reacted site consists of a 5'-phosphate and 3'-hydroxyl group.
  • the large ribozyme is further classified into (1) group I intron RNA catalyzing transesterification at the 5'-splice site by guanosine; (2) group II intron RNA catalyzing self-splicing through a two step reaction via lariat structure; and (3) RNA component of the ribonuclease P that cleaves the tRNA precursor at the 5' site through hydrolysis.
  • small ribozymes have a smaller size (about 40 bp) compared to the large ribozymes and cleave RNAs to generate a 5'-hydroxyl group and a 2'-3' cyclic phosphate.
  • ribozymes inhibiting the expression of the polypeptides described herein can also be constructed based on their sequence information (SEQ ID NO: 24 or 26) and these conventional methods.
  • Ribozymes against B7330N gene inhibit the expression of over-expressed B7330N protein and is thus useful for suppressing the biological activity of the protein. Therefore, the ribozymes are useful in treating or preventing breast cancer.
  • a method for diagnosing cell proliferative disease such as breast cancer using the expression level of the genes described herein as a diagnostic marker is described herein.
  • a method for determining a predisposition to breast cancer in a subject by determining an expression level of the genes of the invention in a patient-derived biological sample, such as tissue sample is also described herein.
  • An alteration, e.g ., an increase in the level of expression of a gene as compared to a normal control level of the gene indicates that the subject may suffer from or is at risk of developing breast cancer.
  • predisposition to breast cancer encompasses a state of a subject of being predisposed to, having a tendency, prevalence, inclination or susceptibility to breast cancer. Moreover, said term also encompasses that a subject is at a risk of acquiring breast cancer.
  • This diagnosing method comprises the steps of: (a) detecting the expression level of the B7330N gene of the present invention; and (b) relating an elevation of the expression level to breast cancer. Likewise, in the method for determining a predisposition to breast cancer the same steps as mentioned before are applied.
  • the expression levels of the B7330N gene in a biological sample can be estimated by quantifying mRNA corresponding to or protein encoded by the B7330N gene. Quantification methods for mRNA are known to those skilled in the art. For example, the levels of mRNAs corresponding to the B7330N gene can be estimated by Northern blotting or RT-PCR. Since the full-length nucleotide sequences of the B7330N genes are shown in SEQ ID NO: 24 or 26, anyone skilled in the art can design the nucleotide sequences for probes or primers to quantify the B7330N gene.
  • the expression level of the B7330N gene can be analyzed based on the activity or quantity of protein encoded by the gene.
  • a method for determining the quantity of the B7330N protein is shown in below.
  • immunoassay methods are useful for the determination of the proteins in biological materials. Any biological materials can be used as the biological sample for the determination of the protein or it's activity so long as the marker gene ( B7330N gene) is expressed in the sample of a breast cancer patient
  • breast duct epithelium can be mentioned as such biological sample.
  • bodily fluids such as blood and urine may be also analyzed.
  • a suitable method can be selected for the determination of the activity of a protein encoded by the B7330N gene according to the activity of a protein to be analyzed.
  • Expression levels of the B7330N gene in a biological sample are estimated and compared with those in a normal sample (e.g ., a sample derived from a non-diseased subject). When such a comparison shows that the expression level of the target gene is higher than those in the normal sample, the subject is judged to be affected with breast cancer.
  • the expression level of a B7330N gene in the biological sample(s) from a normal subject and subject to be diagnosed may be determined at the same time.
  • normal ranges of the expression levels can be determined by a statistical method based on the results obtained by analyzing the expression level of the gene in samples previously collected from a control group. A result obtained by comparing the sample of a subject is compared with the normal range; when the result does not fall within the normal range, the subject is judged to be affected with or is at risk of developing breast cancer.
  • a diagnostic agent for diagnosing cell proliferative disease such as breast cancer
  • the diagnostic agent described herein comprises a compound that binds to a polynucleotide or a polypeptide described herein.
  • an oligonucleotide that hybridizes to the polynucleotide described herein or an antibody that binds to the polypeptide described herein may be used as such a compound.
  • an aptamer such as a RNA, DNA or peptide aptamers may be used.
  • the present method of diagnosing breast cancer may be applied for assessing the efficacy of treatment of breast cancer in a subject
  • a biological sample such as a test cell population
  • the method for assessment can be conducted according to conventional methods of diagnosing breast cancer.
  • biological samples are obtained from the subject at various time points before, during or after the treatment.
  • the expression level of B7330N gene, in the biological sample is then determined and compared to a control level derived, for example, from a reference cell population which includes cells whose state of breast cancer (i.e. , cancerous cell or non-cancerous cell) is known.
  • the control level is determined in a biological sample that has not been exposed to the treatment.
  • control level is derived from a biological sample which contains no cancerous cell
  • a similarity between the expression level in the subject-derived biological sample and the control level indicates that the treatment is efficacious.
  • a difference between the expression level of the B7330N gene in the subject-derived biological sample and the control level indicates a less favorable clinical outcome or prognosis.
  • efficacious refers that the treatment leads to a reduction in the expression of a pathologically up-regulated gene ( B7330N gene) or a decrease in size, prevalence or proliferating potential of breast cancer cells in a subject.
  • a treatment is applied prophylactically, “efficacious” indicates that the treatment retards or prevents occurrence of breast cancer.
  • the assessment of breast cancer can be made using standard clinical protocols.
  • the efficaciousness of a treatment is determined in association with any known method for diagnosing or treating breast cancer.
  • the present method of diagnosing breast cancer may also be applied for assessing the prognosis of a subject with breast cancer by comparing the expression level of B7330N gene in a patient-derived biological sample; such as test cell population, to a control level.
  • the expression level of B7330N gene in a biological sample derived from patients may be measured over a spectrum of disease stages to assess the prognosis of the patient.
  • An increase in the expression level of B7330N gene compared to a normal control level indicates less favorable prognosis.
  • a similarity in the expression level of B7330N gene compared to a normal control level indicates a more favorable prognosis for the patient.
  • proteins encoded by the gene or transcriptional regulatory region of the gene proteins encoded by the gene or transcriptional regulatory region of the gene, compounds can be screened that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene. Such compounds are used as pharmaceuticals for use in treating or preventing breast cancer.
  • This screening method comprises the steps of: (a) contacting a test compound with a polypeptide described herein ; (b) detecting the binding activity between the polypeptide described herein and the test compound; and (c) selecting the compound that binds to the polypeptide described herein
  • All embodiments described herein with respect to the polypeptide, polynucleotide, vectors and/or host cells described herein also pertain to the herein disclosed screening methods, mutatis mutandis, which apply said polypeptide, polynucleotide, vectors and/or host cells.
  • the polypeptide described herein to be used for screening may be a recombinant polypeptide or a protein derived from the nature or a partial peptide thereof.
  • the polypeptide described herein to be contacted with a test compound can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.
  • a method of screening for proteins for example, that bind to the polypeptide of the present invention using the polypeptide of the present invention
  • many methods well known by a person skilled in the art can be used.
  • Such a screening can be conducted by, for example, immunoprecipitation method, specifically, in the following manner.
  • the gene encoding the polypeptide described herein is expressed in host ( e.g. , animal) cells and so on by inserting the gene to an expression vector for foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8.
  • the promoter to be used for the expression may be any promoter that can be used commonly and include, for example, the SV40 early promoter ( Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 83-141 (1982 )), the EF- ⁇ promoter ( Kim et al., Gene 91: 217-23 (1990 )), the CAG promoter ( Niwa et al., Gene 108: 193 (1991 )), the RSV LTR promoter ( Cullen, Methods in Enzymology 152: 684-704 (1987 )) the SR ⁇ promoter ( Takebe et al., Mol Cell Biol 8: 466 (1988 )), the CMV immediate early promoter ( Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9 (1987 )), the SV40 late promoter ( Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982 )), the Adenovirus late promoter ( Kauf
  • the introduction of the gene into host cells to express a foreign gene can be performed according to any methods, for example, the electroporation method ( Chu et al., Nucleic Acids Res 15: 1311-26 (1987 )), the calcium phosphate method ( Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987 )), the DEAE dextran method ( Lopata et al., Nucleic Acids Res 12: 5707-17 (1984 ); Sussman and Milman, Mol Cell Biol 4: 1641-3 (1984 )), the Lipofectin method ( Derijard B, Cell 76: 1025-37 (1994 ); Lamb et al., Nature Genetics 5: 22-30 (1993 ): Rabindran et al., Science 259: 230-4 (1993 )) and so on.
  • the electroporation method Chu et al., Nucleic Acids Res 15: 1311-26 (1987 )
  • the calcium phosphate method Chen and Okayama, Mol Cell
  • the polypeptide described herein can be expressed as a fusion protein comprising a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide described herein .
  • a commercially available epitope-antibody system can be used ( Experimental Medicine 13: 85-90 (1995 )).
  • Vectors which can express a fusion protein with, for example, ⁇ -galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available.
  • a fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the polypeptide described herein: by the fusion is also reported.
  • Epitopes such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the polypeptide described herein ( Experimental Medicine 13: 85-90 (1995 )).
  • an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent
  • the immune complex consists of the polypeptide of the present invention, a polypeptide comprising the binding ability with the polypeptide, and an antibody.
  • Immunoprecipitation can be also conducted using antibodies against the polypeptide of the present invention, besides using antibodies against the above epitopes, which antibodies can be prepared as described above.
  • an immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody.
  • an immune complex can be formed in the same manner as in the use of the antibody against the polypeptide of the present invention, using a substance specifically binding to these epitopes, such as glutathione-Sepharose 4B.
  • Immunoprecipitation can be performed by following or according to, for example, the methods in the literature ( Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988 )).
  • SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Since the protein bound to the polypeptide described herein is difficult to detect by a common staining method, such as Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35 S-methionine or 35 S-cysteine, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
  • a common staining method such as Coomassie staining or silver staining
  • a protein binding to the polypeptide described herein can be obtained by preparing a cDNA library from cells, tissues, organs (for example, tissues such as breast cancer cell lines and normal human placenta, pancreas, stomach, trachea, mammary gland and bone marrow.), or cultured cells ( e.g.
  • a phage vector e.g. , ZAP
  • the polypeptide described herein may be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to the polypeptide described herein, or a peptide or polypeptide (for example, GST) that is fused to the polypeptide described herein. Methods using radioisotope or fluorescence and such may be also used.
  • a two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references ⁇ Dalton and Treisman, Cell 68: 597-612 (1992 )", “ Fields and Sternglanz, Trends Genet 10: 286-92 (1994 )”).
  • the polypeptide described herein is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells.
  • a cDNA library is prepared from cells expected to express a protein binding to the polypeptide of the invention, such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region.
  • the cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the polypeptide of the invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable).
  • a protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E . coli and expressing the protein.
  • reporter gene for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
  • a compound binding to the polypeptide described herein can also be screened using affinity chromatography.
  • the polypeptide of the invention may be immobilized on a carrier of an affinity column, and a test compound, containing a protein capable of binding to the polypeptide of the invention, is applied to the column.
  • a test compound herein may be, for example, cell extracts, cell lysates, etc . After loading the test compound, the column is washed, and compounds bound to the polypeptide described herein can be prepared.
  • test compound When the test compound is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.
  • a biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound compound described herein
  • a biosensor When such a biosensor is used, the interaction between the polypeptide of the invention and a test compound can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide of the invention and a test compound using a biosensor such as BIAcore.
  • a method of screening for a compound for use in treating or preventing breast cancer using the polypeptide described herein comprising the steps as follows:
  • the B7330N protein of the present invention have the activity of promoting cell proliferation of breast cancer cells, a compound which inhibits this activity of this protein of the present invention can be screened using this activity as an index.
  • Any polypeptides can be used for screening so long as they comprise the biological activity of the B7330N protein.
  • biological activity includes cell-proliferating activity of the human B7330N protein.
  • a human B7330N protein can be used and polypeptides functionally equivalent to these proteins can also be used.
  • polypeptides may be expressed endogenously or exogenously by cells.
  • the present invention is based in part on the discovery of a novel glycosylation of B7330N protein, which is involved in proliferation of cancer cells, as detailed below.
  • B7330 plays a role in autocrine regulation of cell growth through the activation of signal transduction for cell proliferation ( Fig. 5a, 5b ).
  • N476A mutant which was replaced 476N (asparagine) with A (alanine)
  • no glycosylation was observed ( Fig. 3b ).
  • Exogenous wild-type B7330N was secreted into culture medium, whereas the N476A protein was not detected in culture medium, suggesting glycosylation on Asn-476 of B7330N protein is necessary for secretion ( Fig. 3c ).
  • cells expressing wild type of B7330N grew much faster than cells transfected with N476A ( Fig. 8b ). Accordingly, cell growth of breast cancer may be supressed by inhibition of the glycosylation of B7330N.
  • a method of screening for a compound for use in treating or preventing breast cancer using the polypeptide described herein comprises the steps as follows:
  • said glycosylation level is that of asparagines 476 of the polypeptide of the present invention, in particular it is the glycosylation level of asparagine 476 of the amino acid sequence of SEQ ID NO: 25 or a homologous portion thereof.
  • the skilled person is readily in a position to determine in a polypeptide of the present invention the position corresponding to position 476 of the amino acid sequence of SEQ ID NO: 25.
  • These methods are practiced by contacting a cell expressing B7330N polypeptide or functional equivalent thereof having glycosylation site, or a polypeptide itself with one or more candidate compounds, and detecting glycosylation level of the contacted B7330N or the functional equivalent.
  • a compound that modulates glycosylation level of the B7330N or functional equivalent is thereby identified.
  • the term “functionally equivalent” also means herein that the subject protein has the same or substantially the same glycosylation level as B7330N.
  • the protein or a partial amino acid of the protein which includes glycosylation site are catalyzed the glycosylation. Whether a subject protein has the target activity can be determined herein. Namely, the glycosylation level of the B7330N protein can be detected by contacting a polypeptide with a test compound under conditions suitable for glycosylation of the protein.
  • glycosylation level of a B7330N polypeptide can be determined herein methods known in the art. For example, glycosylation of the polypeptide may be detected by comparing the molecular weight Molecular weight of a glycosylated protein is larger than that of predicted size caluculated from the amino acid sequence of the polypeptide by addition of glycoside chain. Furthermore, when the molecular weight of glycosylated protein might be reduced by glycosidase treatment, it was confirmed that the difference of the molecular is caused by addition of glycoside chain. Methods for estimating a molecular weight of a protein are well known.
  • radiolabeled donor for glycosylation may be used for detection the addition of glycoside chain to the polypeptide.
  • Transfer of the radiolabel to the B7330N protein can be detected, for example, by SDS-PAGE electrophoresis and fluorography.
  • the B7330N peptides can be separated from the glycosyl donor by filtration, and the amount of radiolabel retained on the filter quantitated by scintillation counting.
  • Other suitable labels that can be attached to glycosyl donor such as chromogenic and fluorescent labels, and methods of detecting transfer of these labels to the B7330N protein, are known in the art.
  • glycosylation level of B7330N can be determined reagents that selectively recognize glycosylated level of the polypeptide. For example, after incubation of the B7330N polypeptide and candidate compound, under the condition capable of glycosylation of the polypeptide, the glycosylation level of the polypeptide can be detected by immunological method. Any immunological techniques using an antibody recognizing glycosylated polypeptide can be used for the detection. For example, an antibody against glycosylated polypeptide is commercial available. ELISA or Immunoblotting with antibodies recognizing glycosylated polypeptide can be used for the present invention.
  • glycosylated protein can be detected using reagents that selectively bind glycoside chain with high affinity.
  • reagents are known in the art or can be determined by screening assays known in the art.
  • lectins are well known as glycoside chain specific probe.
  • Lectin reagent conjugated with detectable label such as alkaline-phosphatase is also cmmercialy available.
  • Glycosylation level of polypeptide in a cell may be estimated by separation of cell lysate.
  • SDS-polyacrylamide gel can be used as the separation of the polypeptide.
  • the polypeptide separated in the gels is transferred to nitrocellulose membranes for immunoblotting analysys.
  • the compound isolated by this screening is a candidate for agonists or antagonists of the polypeptide described herein.
  • agonist refers to molecules that activate the function of the polypeptide of the present invention by binding thereto.
  • antagonist refers to molecules that inhibit the function of the polypeptide described herein by binding thereto.
  • a compound isolated by this screening is a candidate for compounds which inhibit the in vivo interaction of the polypeptide of the present invention with molecules (including DNAs and proteins).
  • the biological activity to be detected in the present method is cell proliferation
  • it can be detected, for example, by preparing cells which express the polypeptide of the present invention, culturing the cells in the presence of a test compound, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring the colony forming activity as described in the Examples.
  • Cells expressing at least one of the B7330N include, for example, cell lines established from breast cancers; such cells can be used for the above screening of the present invention (e.g. , HBC4, HBC5, MCF-7, MDA-MB-231, YMB1, SKBR3, T47D, BT-20, HCC1500 or MDA-MB-453. etc .).
  • the expression level can be estimated by methods well known to one skilled in the art.
  • a compound that reduces the expression level of B7330N can be selected as candidate agents to be used for the treatment or prevention of breast cancer.
  • the screening method described herein may comprise the following steps:
  • Suitable reporter genes and host cells are well known in the art.
  • the reporter construct required for the screening can be prepared by using the transcriptional regulatory region of a marker gene.
  • a reporter construct can be prepared by using the previous sequence information.
  • a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library based on the nucleotide sequence information of the marker gene.
  • supports that may be used for binding proteins include insoluble polysaccharides, such as agarose, cellulose and dextran; and synthetic resins, such as polyacrylamide, polystyrene and silicon; preferably commercial available beads and plates ( e.g ., multi-well plates, biosensor chip, etc.) prepared from the above materials may be used. When using beads, they may be filled into a column.
  • insoluble polysaccharides such as agarose, cellulose and dextran
  • synthetic resins such as polyacrylamide, polystyrene and silicon
  • beads When using beads, they may be filled into a column.
  • binding of a protein to a support may be conducted according to routine methods, such as chemical bonding and physical adsorption.
  • a protein may be bound to a support via antibodies specifically recognizing the protein.
  • binding of a protein to a support can be also conducted by means of avidin and biotin.
  • the binding between proteins is carried out in buffer, for example, but are not limited to, phosphate buffer and Tris buffer, as long as the buffer does not inhibit the binding between the proteins.
  • a biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound protein described herein.
  • the interaction between the proteins can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia).
  • B7330N polypeptide may be labeled, and the label of the bound protein may be used to detect or measure the bound protein. Specifically, after pre-labeling one of the proteins, the labeled protein is contacted with the other protein in the presence of a test compound, and then bound proteins are detected or measured according to the label after washing.
  • Labeling substances such as radioisotope (e.g. , 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, 131 I), enzymes (e.g. , alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, ⁇ -glucosidase), fluorescent substances (e.g. , fluorescein isothiosyanete (FTTC), rhodamine) and biotin/avidin, may be used for the labeling of a protein in the present method When the protein is labeled with radioisotope, the detection or measurement can be carried out by liquid scintillation.
  • radioisotope e.g. , 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, 131 I
  • enzymes e.g. , alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, ⁇ -glu
  • proteins labeled with enzymes can be detected or measured by adding a substrate of the enzyme to detect the enzymatic change of the substrate, such as generation of color, with absorptiometer. Further, in case where a fluorescent substance is used as the label, the bound protein may be detected or measured using fluorophotometer.
  • the antibody is preferably labeled with one of the labeling substances mentioned above, and detected or measured based on the labeling substance.
  • the antibody against the B7330N polypeptide or actin may be used as a primary antibody to be detected with a secondary antibody that is labeled with a labeling substance.
  • the antibody bound to the protein in the screening of the present invention may be detected or measured using protein G or protein A column.
  • test compound for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds and natural compounds can be used in the screening methods of the present invention.
  • the test compound described herein can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-compound” library method and (5) synthetic library methods using affinity chromatography selection.
  • a compound isolated by the screening methods described herein is a candidate for drugs which inhibit the activity of the polypeptide described herein, for use in treating or preventing diseases attributed to, for example, cell proliferative diseases, such as breast cancer.
  • compositions for use in treating or preventing breast cancer are provided.
  • compositions for use in treating or preventing breast cancer comprising any of the compounds selected by the screening methods described above are also disclosed.
  • the isolated compound When administrating a compound isolated by the screening methods described herein as a pharmaceutical for humans or other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, chimpanzees, for treating a cell proliferative disease (e.g. , breast cancer) the isolated compound can be directly administered or can be formulated into a dosage form using known pharmaceutical preparation methods.
  • the drugs can be taken orally, as sugarcoated tablets, capsules, elixirs and microcapsules; or non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid.
  • the compounds can be mixed with pharmacologically acceptable carriers or medium, specifically, sterilized water, physiological saline, plant-oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders and such, in a unit dose form required for generally accepted drug implementation.
  • pharmacologically acceptable carriers or medium specifically, sterilized water, physiological saline, plant-oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders and such, in a unit dose form required for generally accepted drug implementation.
  • the amount of active ingredients in these preparations makes a suitable dosage within the indicated range acquirable.
  • additives that can be mixed to tablets and capsules are, binders such as gelatin, corn starch, tragacanth gum and arabic gum; excipients such as crystalline cellulose; swelling agents such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin; flavoring agents such as peppermint, Gaultheria adenothrix oil and cherry.
  • a liquid carrier such as oil, can also be further included in the above ingredients.
  • Sterile composites for injections can be formulated following normal drug implementations using vehicles such as distilled water used for injections.
  • Physiological saline, glucose, and other isotonic liquids including adjuvants can be used as aqueous solutions for injections.
  • adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride
  • Suitable solubilizers such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (TM) and HCO-50.
  • Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizers and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol, phenol; and an anti-oxidant
  • the prepared injection may be filled into a suitable ampule.
  • Methods well known to one skilled in the art may be used to administer the inventive pharmaceutical compound to patients, for example as intraarterial, intravenous, percutaneous injections and also as intranasal, intramuscular or oral administrations.
  • the dosage and method of administration vary according to the body-weight and age of a patient and the administration method; however, one skilled in the art can routinely select them. If said compound is encodable by a DNA, the DNA can be inserted into a vector for gene therapy and the vector administered to perform the therapy.
  • the dosage and method of administration vary according to the body-weight, age, and symptoms of a patient but one skilled in the art can select them suitably.
  • the dose of a compound that binds with the polypeptide of the present invention and regulates its activity is about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult (weight 60 kg).
  • compositions for use in treating or preventing breast cancer comprising active ingredients that inhibits the expression of B7330N gene as described herein.
  • active ingredients include antisense polynucleotides, siRNAs or ribozymes against the B7330N gene or derivatives, such as expression vector, of the antisense polynucleotides, siRNAs or ribozymes.
  • active ingredients can be made into an external preparation, such as a liniment or a poultice, by mixing with a suitable base material which is inactive against the derivatives. Also, as needed, they can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers and such. These can be prepared according to conventional methods.
  • the active ingredient is given to the patient by directly applying onto the ailing site or by injecting into a blood vessel so that it will reach the site of ailment.
  • a mounting medium can also be used to increase durability and membrane-permeability. Examples of mounting medium include liposome, poly-L-lysine, lipid, cholesterol, lipofectine or derivatives of these.
  • compositions of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts.
  • a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
  • a pharmaceutical composition for use in treating or preventing breast cancer comprising an antibody against a polypeptide encoded by the B7330N gene or fragments of the antibody that bind to the polypeptide is also described herein.
  • the dose of an antibody or fragments thereof for treating or preventing breast cancer is about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult (weight 60 kg).
  • Therapeutic compounds for use in treating or preventing breast cancer in a subject are also described herein.
  • Therapeutic compounds are administered prophylactically or therapeutically to subject suffering from or at risk of (or susceptible to) developing breast cancer. Such subjects are identified using standard clinical methods or by detecting an aberrant expression level or activity of B7330N. Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • the therapeutic method includes decreasing the expression or function of B7330N gene.
  • the subject is treated with an effective amount of a compound, which decreases the over-expressed gene (B7330N gene) in the subject
  • Administration can be systemic or local.
  • Therapeutic compounds include compounds that decrease the expression level of such gene endogenously existing in the breast cancerous cells (i.e. , compounds that down-regulate the expression of the over-expressed gene).
  • Administration of such therapeutic compounds counter the effects of aberrantly-over expressed gene in the subject's cells and are expected to improve the clinical condition of the subject
  • Such compounds can be obtained by the screening method described above.
  • B7330N gene may be also inhibited in any of several ways known in the art including administering to the subject a nucleic acid that inhibits or antagonizes the expression of the gene.
  • Antisense oligonucleotides, siRNA or ribozymes which disrupts expression of the gene can be used for inhibiting the expression of the gene.
  • Antisense oligonucleotides, siRNA or ribozymes which can be used for inhibiting the expression of B7330N gene are described herein above.
  • antisense-oligonucleotides corresponding to the nucleotide sequence of B7330N gene can be used to reduce the expression level of the B7330N gene.
  • the antisense-oligonucleotides described herein may act by binding to any of the polypeptides encoded by the B7330N gene, or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the gene, promoting the degradation of the mRNAs, and/or inhibiting the expression of proteins encoded by the gene, and finally inhibiting the function of the B7330N proteins.
  • An antisense-oligonucleotides and derivatives thereof can be made into an external preparation, such as a liniment or a poultice, by mixing with a suitable base material which is inactive against the derivative and used in the method for use in treating or preventing breast cancer described herein.
  • the nucleic acids that inhibit a gene product of over-expressed gene also include small interfering RNAs (siRNA) comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid of the nucleotide sequence encoding the B7330N gene.
  • siRNA small interfering RNAs
  • Standard techniques of introducing siRNA into the cell can be used in the treatment or prevention of the present invention, including those in which DNA is a template from which RNA is transcribed.
  • the siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g. , a hairpin, to suppress gene expression of a cell with up-regulated expression of the B7330N gene. Binding of the siRNA to the B7330N gene transcript in the target cell results in a reduction of B7330N protein production by the cell.
  • the nucleic acids that inhibit a gene product of over-expressed gene also include ribozymes against the over-expressed gene ( B7330N gene).
  • B7330N gene ribozymes against the over-expressed gene
  • an antibody against the polypeptide described herein for use in treating or preventing a cell proliferative disease, such as breast cancer is described herein, wherein a pharmaceutically effective amount of an antibody against the polypeptide described herein is administered. Since the expression of the B7330N protein are up-regulated in breast cancer cells and the suppression of the expression of these proteins leads to the decrease in cell proliferating activity, it is expected that cell proliferative diseases can be treated or prevented by binding the antibody and these proteins.
  • an antibody against the polypeptide described herein is administered at a dosage sufficient to reduce the activity of the protein described herein which is in the range of 0.1 to about 250 mg/kg per day.
  • the dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day.
  • an antibody binding to a cell surface marker specific for tumor cells can be used as a tool for drug delivery.
  • the antibody conjugated with a cytotoxic agent is administered at a dosage sufficient to injure tumor cells.
  • Another aspect described herein comprises the agent which inhibits the glycosylation of asparagine 476 of an amino acid sequence of a polypeptide described herein in particular of the amino acid sequence of a polypeptide of SEQ ID NO: 25 for use in treating or preventing breast cancer.
  • a pharmaceutical composition comprising the B7330N protein or an immunologically active fragment thereof, or a polynucleotide encoding the protein or fragment thereof for use in inducing anti-tumor immunity is also described herein.
  • the B7330N protein or the immunologically active fragments thereof are useful as vaccines against cell proliferative diseases such as breast cancer.
  • the proteins or fragments thereof may be administered in a form bound to the T cell receptor (TCR) or presented by an antigen presenting cell (APC), such as macrophage, dendritic cell (DC), or B-cells. Due to the strong antigen presenting ability of DC, the use of DC is most preferable among the APCs.
  • TCR T cell receptor
  • APC antigen presenting cell
  • DC dendritic cell
  • B-cells B-cells. Due to the strong antigen presenting ability of DC, the use of DC is most preferable among the APCs.
  • Vaccine against cell proliferative disease refers to a substance that has the function to induce anti-tumor immunity upon inoculation into animals.
  • anti-tumor immunity includes immune responses such as follows:
  • the protein when a certain protein induces any one of these immune responses upon inoculation into an animal, the protein is decided to have anti-tumor immunity inducing effect.
  • the induction of the anti-tumor immunity by a protein can be detected by observing in vivo or in vitro the response of the immune system in the host against the protein.
  • cytotoxic T lymphocytes For example, a method for detecting the induction of cytotoxic T lymphocytes is well known.
  • a foreign substance that enters the living body is presented to T cells and B cells by the action of antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • T cells that respond to the antigen presented by APC in antigen specific manner differentiate into cytotoxic T cells (or cytotoxic T lymphocytes; CTLs) due to stimulation by the antigen, and then proliferate (this is referred to as activation of T cells). Therefore, CTL induction by a certain peptide can be evaluated by presenting the peptide to T cell by APC, and detecting the induction of CTL.
  • APC has the effect of activating CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells. Since CD4+ T cells and CD8+ T cells are also important in anti-tumor immunity, the anti-tumor immunity inducing action of the peptide can be evaluated using the activation effect of these cells as indicators.
  • a method for evaluating the inducing action of CTL using dendritic cells (DCs) as APC is well known in the art.
  • DC is a representative APC having the strongest CTL inducing action among APCs.
  • the test polypeptide is initially contacted with DC, and then this DC is contacted with T cells. Detection of T cells having cytotoxic effects against the cells of interest after the contact with DC shows that the test polypeptide has an activity of inducing the cytotoxic T cells.
  • Activity of CTL against tumors can be detected, for example, using the lysis of 51 Cr-labeled tumor cells as the indicator.
  • the method of evaluating the degree of tumor cell damage using 3 H-thymidine uptake activity or LDH (lactose dehydrogenase)-release as the indicator is also well known.
  • peripheral blood mononuclear cells may also be used as the APC.
  • the induction of CTL is reported that it can be enhanced by culturing PBMC in the presence of GM-CSF and IL-4.
  • CTL has been shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.
  • KLH keyhole limpet hemocyanin
  • test polypeptides confirmed to possess CTL inducing activity by these methods are polypeptides having DC activation effect and subsequent CTL inducing activity. Therefore, polypeptides that induce CTL against tumor cells are useful as vaccines against tumors. Furthermore, APC that acquired the ability to induce CTL against tumors by contacting with the polypeptides are useful as vaccines against tumors. Furthermore, CTL that acquired cytotoxicity due to presentation of the polypeptide antigens by APC can be also used as vaccines against tumors. Such therapeutic methods for tumors using anti-tumor immunity due to APC and CTL are referred to as cellular immunotherapy.
  • the induction of anti-tumor immunity by a polypeptide can be confirmed by observing the induction of antibody production against tumors. For example, when antibodies against a polypeptide are induced in a laboratory animal immunized with the polypeptide and when growth of tumor cells is suppressed by those antibodies, the polypeptide can be determined to have an ability to induce anti-tumor immunity.
  • Anti-tumor immunity is induced by administering the vaccine of this invention, and the induction of anti-tumor immunity enables treatment and prevention of cell proliferating diseases, such as breast cancers.
  • Therapy against cancer or prevention of the onset of cancer includes any of the steps, such as inhibition of the growth of cancerous cells, involution of cancer and suppression of occurrence of cancer. Decrease in mortality of individuals having cancer, decrease of tumor markers in the blood, alleviation of detectable symptoms accompanying cancer and such are also included as the effect of therapy or prevention of cancer.
  • Such therapeutic and preventive effects are preferably statistically significant For example, in observation, at a significance level of 5% or less, wherein the therapeutic or preventive effect of a vaccine against cell proliferative diseases is compared to a control without vaccine administration.
  • Student's t-test, the Mann-Whitney U-test or ANOVA may be used for statistical analysis.
  • the above-mentioned protein having immunological activity or a vector encoding the protein may be combined with an adjuvant.
  • An adjuvant refers to a compound that enhances the immune response against the protein when administered together (or successively) with the protein having immunological activity.
  • adjuvants include cholera toxin, salmonella toxin, alum and such, but are not limited thereto.
  • the vaccine of this invention may be combined appropriately with a pharmaceutically acceptable carrier. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid and such.
  • the vaccine may contain as necessary, stabilizers, suspensions, preservatives, surfactants and such.
  • the vaccine is administered systemically or locally. Vaccine administration may be performed by single administration or boosted by multiple administrations.
  • tumors can be treated or prevented, for example, by the ex vivo method. More specifically, PBMCs of a subject receiving treatment or prevention therapy are collected, the cells are contacted with the polypeptide ex vivo, and following the induction of APC or CTL, the cells may be administered to the subject.
  • APC can be also induced by introducing a vector encoding the polypeptide into PBMCs ex vivo.
  • APC or CTL induced in vitro can be cloned prior to administration. By cloning and growing cells having high activity of damaging target cells, cellular immunotherapy can be performed more effectively.
  • APC and CTL isolated in this manner may be used for cellular immunotherapy not only against individuals from whom the cells are derived, but also against similar types of tumors from other individuals.
  • a pharmaceutical composition for use in treating or preventing a cell proliferative disease, such as breast cancer comprising a pharmaceutically effective amount of the B7330N polypeptide is described herein.
  • the pharmaceutical composition may be used for raising anti tumor immunity.
  • the normal expression of B7330N is restricted to placenta, pancreas, stomach, trachea, mammary gland and bone marrow. Therefore, suppression of this gene may not adversely affect other organs.
  • the B7330N polypeptides are preferable for use in treating cell proliferative disease, especially breast cancers.
  • peptide fragments of proteins specifically expressed in cancerous cells were revealed to induce immune response against the cancer
  • peptide fragments of B7330N can also be used in a pharmaceutical composition for use in treating or preventing cell proliferative diseases such as breast cancers.
  • the polypeptide or fragment thereof described herein is administered at a dosage sufficient to induce anti-tumor immunity, which is in the range of 0.1 mg to 10 mg, preferably 0.3 mg to 5 mg, more preferably 0.8 mg to 1.5 mg.
  • the administrations are repeated. For example, 1 mg of the peptide or fragment thereof may be administered 4 times in every two weeks for inducing the anti-tumor immunity.
  • polynucleotides encoding B7330N, or fragments thereof may be used for raising anti tumor immunity. Such polynucleotides may be incorporated in an expression vector to express B7330N, or fragments thereof in a subject to be treated.
  • a pharmaceutical composition comprising the polynucleotides encoding B7330N, or fragments thereof for use in inducing anti-tumor immunity is described herein, wherein said pharmaceutical composition is administered to a subject suffering or being at risk of developing cell proliferative diseases such as breast cancer.
  • Human-breast cancer cell lines HBL-100, HCC1937, MCF-7, MDA-MB-435s, YMB1, SKBBR3, T47D, BT-20, BT-474, BT-549, HCC1143, HCC1500, HCC1599, MDA-MB-157, MDA-MB4S3, OUCB-F, ZR-75-1 and COS-7 cell lines are purchased from American Type Culture Collection (ATCC) and are cultured under their respective depositors' recommendation.
  • ATCC4 American Type Culture Collection
  • HBC4 and MDA-MB-231 cells lines are kind gifts from Dr. Yamori of Molecular Pharmacology, Cancer Chemotherapy Centre of the Japanese Foundation for Cancer Research. All cells were cultured in appropriate media; i.e.
  • RPMI-1640 (Sigma, St. Louis, MO) for HBC4, HBC5, T47D, YMB1, OUCB-F, ZR-75-1, BT-549, HCC1143, HCC1500, HCC1599 and HCC1937 (with 2mM L-glutamine); Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) for BT474, HBL100, COS7; EMEM (Sigma) with 0.1mM essential amino acid (Roche), 1mM sodium pyruvate (Roche), 0.01 mg/ml Insulin (Sigma) for BT-20 and MCF-7; McCoy (Sigma) for SKBR3 (with 1.5mM L-glutamine); L-15 (Roche) for MDA-MB-231, MDA-MB-157, MDA-MB453 and MDA-MB-43Ss.
  • MDA-MB-231 and MDA-MB-435s cells were maintained at 37°C an atmosphere of humidified air without CO 2 .
  • Other cell lines were maintained at 37°C an atmosphere of humidified air with 5% CO 2 .
  • Clinical samples (breast cancer and normal breast duct) were obtained from surgical specimens, concerning which all patients had given informed consent.
  • RNA samples were purified from each sample of laser-microdissected cells, and T7-based RNA amplification was carried out to obtain adequate quantities of RNA for microarray experiments. Aliquots of amplified RNA from breast cancer cells and the normal breast ductal cells were labeled by reverse transcription with Cy5-dCTP and Cy3-dCTP, respectively (Amersham Biosciences, Buckinghamshire, UK).
  • Hybridization, washing, and detection were carried out as described previously ( Ono K, et al., (2000) Cancer Res., 60, 5007-11 ).
  • the overall expression patterns of the 27,648 genes on the microarray were screened to select those with expression ratios >3.0 that were present in >50% of i) all of 77 premenopausal breast cancer cases, ii) 69 invasive ductal carcinomas, iii) 31 well-, iv) 14 moderately-, or v) 24 poorly-differentiated lesions, respectively.
  • B7330N Because its expression ratio was greater than 3.0 in more than 30% of the informative breast cancer cases.
  • RNA was extracted total RNA from each population of laser-captured cells and then performed T7-based amplification and reverse transcription as described previously ( Kitahara O, et al., (2001) Cancer Res., 61, 3544-9 ).
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • the PCR primer sequences were 5'-CGACCACTTTGTCAAGCTCA-3' (SEQ ID NO; 1) and 5'- GGTTGAGCACAGGGTACTTTATT-3' (SEQ ID NO; 2) for GAPDH; and 5'-GAGTCCAGGTAAGTGAATCTGTCC-3' (SEQ ID NO; 3) and 5'-ATTTCCACCGAGACCTCTCATC-3' (SEQ ID NO; 4) for B7330N.
  • RNAs were extracted from all breast cancer cell lines using RNeasy kit (QIAGEN) according to the manufacturer's instructions. After treatment with DNase I (Nippon Gene, Osaka, Japan), mRNA was isolated with mRNA purification kit (Amersham Biosciences) following the manufacturer's instructions. A 1- ⁇ g aliquot of each mRNA, along with polyA(+) RNAs isolated from normal adult human breast (BioChain), lung, heart, liver, kidney, bone marrow (BD, Clontech, Palo Alto, CA), were separated on 1% denaturing agarose gels and transferred to nylon membranes (Breast cancer-Northern blots).
  • Specific probes for B7330N (502 bp) was prepared by RT-PCR using the following primer set; 5'-GAGTCCAGGTAAGTGAATCTGTCC-3' (SEQ ID NO; 3) and 5'- ATTTCCACCGAGACCTCTCATC-3' (SEQ ID NO; 4) and is radioactively labeled with megaprime DNA labeling system (Amersham Bioscience).
  • B7330N expression vectors For constructing of B7330N expression vectors, the entire coding sequence of B7330N cDNA was amplified by the PCR using KOD-Plus DNA polymerase (Toyobo, Osaka, Japan) and the following primers; forward, 5'-CGGAATTCATGAGGCTCCTCCGCAG-3' (SEQ ID NO; 5), (underline indicates EcoR I site), reverse, 5'-CCGCTCGAGGACAAAGAGCCACAACTGATG-3'. (SEQ ID NO; 6) (underline indicates Xho I site). The PCR products were inserted into the EcoR I and Xho I sites of pCAGGS-HA expression vector.
  • KOD-Plus DNA polymerase Toyobo, Osaka, Japan
  • primers forward, 5'-CGGAATTCATGAGGCTCCTCCGCAG-3' (SEQ ID NO; 5), (underline indicates EcoR I site), reverse, 5'-CCGCTCGAGGACAAAGAGCCACAACTGA
  • B7330N-HA-transfected COS7 cells were incubated with a rat anti-HA antibody (Roche) at 1:1000 dilution. After washing with PBS(-), transfected-cells were stained by an Alexa488-conjugated anti-rat secondary antibody (Molecular Probe) at 1:1000 dilution. Nuclei were counter-stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI). Fluorescent images were obtained under a TCS SP2 AOBS microscope (Leica, Tokyo, Japan).
  • Plasmids designed to express two fragments of B7336N (35-239 a.a.) with His-tagged epitope at their N-terminus and C-terminus was prepared using pET28 vector (Novagen, Madison, WI).
  • the recombinant peptide was expressed in Escherichia coli, BL21 codon-plus strain (Stratagene, La Jolla, CA), and purified using Ni-NTA resin agarose (Qiagen) according to the supplier's protocols.
  • the purified recombinant protein was immunized into rabbits.
  • the immune sera were purified on affinity columns using a recombinant protein (35-239 a.a.) according to standard methodology.
  • Affinity-purified anti-B7330N antibodies were used for western blotting, immunocytostaining and immunohistochemical staining as described below.
  • HMEC human mammary gland epitherial cell
  • lysis buffer as described above.
  • the amount of total protein was estimated by protein assay kit (Bio-Rad, Hercules, CA). and then mixed with SDS-sample buffer and boiled before loading at 10% SDS-PAGE gel as described above. After electrophoresis, the proteins were blotted onto nitrocellulose membrane (GE Healthcare). Membranes including proteins were blocked by blocking solution and incubated with anti-B7330N polyclonal antibody for detection of endogenous B7330N protein. Finally the membrane was incubated with HRP conjugated secondary antibody and protein bands were visualized by ECL detection reagents (GE Healthcare). Beta-actin was examined to serve as a loading control.
  • siRNA expression vector against B7330N (psiU6BX-B7330N) was prepared by cloning of double-stranded oligonucleotides in Table 1 into the Bbs I site in the psiU6BX3.0 vector.
  • Control plasmids, psiU6BX-Mock was prepared by of Bsi I and Hind III of multiple cloning site in the psiU6BX3.0 vector, respectively.
  • Table-1 The siRNA expression vector against B7330N (psiU6BX-B7330N) was prepared by cloning of double-stranded oligonucleotides in Table 1 into the Bbs I site in the psiU6BX3.0 vector.
  • Human breast cancer cells lines T47D or BT-20 was plated onto 15-cm dishes (4x10 6 cells/dish) and transfected with 16 ⁇ g of each psiU6BX-Mock as negative controls and psiU6BX-B7330N using FuGENE6 reagent according to the supplier's recommendations (Roche). 24 hour after transfection, cells are re-seeded again for colony formation assay (2x10 6 cells/10 cm dish), RT-PCR (2x10 6 cells / 10 cm dish) and MTT assay (2x10 6 cells / well).
  • transfectants expressing siRNAs using T47D or BT-20 cell lines were grown for 28 days in selective media containing neomycin, respectively. After fixation with 4% paraformaldehyde, transfected cells were stained with Giemsa solution to assess colony formation. MTT assays were performed to quantify cell viability. After 10 days of culture in the neomycin-containing medium, MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma) was added at a concentration of 0.5 mg/ml.
  • the full length-B7330N and N476A mutant expression vectors were transfected into NIH3T3 cells using FLTGENE6 as describe above. Transfected cells were incubated in the culture medium containing 0.9 mg/ml of geneticin (G418) (Invitrogen). Clonal NIH3T3 cells were subcloned by limiting dilution. Expression and subcellular localization of HA-tagged B7330N were assessed by western blot analysis and immunocytochemistiy using anti-HA monoclonal antibody, respectively. Eventually, several clones were established and designated as WT-B7330N and N476A-B7330N.
  • B7330N-, N476A-, or N611A-transfected COS7 cells were maintained in serum-free medium for 48hours after transfection and then cultured for 4days.
  • B7330N protein in cell lysates and condensed culture media were also detected by rat anti-HA-pAb and anti-rat IgG-HRP.
  • ⁇ -actin pAb (1:2000 dilution) served as a loading control for proteins (clone AC-15, Sigma-Aldrich, MO).
  • cell lysates were also initially prepared as described above without including protease cocktail inhibitor III. Each of 10 ⁇ l cell lysate was mixed with 10 ⁇ l of lysis buffer and 1 U of N-glycosidase F (Calbiochem, San Diego, CA), then incubated for 1 hour at 37 °C. The reaction was boiled with the SDS sample buffer.
  • B7330N-transfected COS7 cells were maintained in FCS-free medium for two days, and then parental COS7 cells were cultivated with or without B7330N in medium to confirm autocrine stimulation of cell growth for 4days. Effects of B7330N on cell growth were monitored by counting cells with a hemocytometer and MTT assay as described above. The cells (5x10 3 cells/well) were cultured in DMEM containing 0.1% FCS.
  • the GALNT6 gene encodes a 622 amino acids protein which is capable of glycosylating fibronectin peptide in vitro and is expressed in a fibroblast cell line, indicating that it may be involved in the synthesis of oncofetal fibronectin.
  • SMART and PFAM computer prediction shows GALNT6 contains signal peptide, Glycos_transf_2 motif in 180 to 370 residues, and RICIN motif in 496 to 622 residues.
  • B7330N To examine the characterization of B7330N, we investigated the sub-cellular localization of these gene products in mammalian cells. Firstly, when we transiently transfected plasmids expressing B7330N protein (pCAGGS-B7330N-HA) into COS7 cells, immunocytochemical analysis with anti-HA-tag antibody reveals that exogenous B7330N protein was appeared as a granulous pattern in secretion vesicles in all transfected-COS7 cells for 72 hours after transfection ( Fig. 2a ).
  • the molecular weight of the B7330N products estimated by western blot analysis was found to be larger than that of the predicted size calculated from the cDNA sequences ( Fig. 2b ). Since the primary amino acid sequence of B7330N contains two predicted N-linked glycosylation consensus sequences, to confirm whether these two larger B7330N products were glycosylation, we initially treated the protein with N-glycosydase. As a result, the largest B7330N protein was disappeared in cell lysate, indicating that this protein was glycosylated in mammalian cells ( Fig. 3a ).
  • RNAi mammalian vector-based RNA interference
  • exogenous pCAGGS-B7330N-HA protein was immunoprecipitated with an anti-HA antibody
  • a western-blot analysis using an anti-HA antibody revealed exogenous B7330N protein in the form a band that corresponded 2-fold of the predicted Molecular mass.
  • pCAGGS-B7330N-HA and pcDNA3.1-B7330N-myc were co-transfected into COS-7 cells and co-immunoprecipitated using anti-HA or anti-myc antibody, respectively. As shown in Fig.
  • B7330N designed to UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 6, GALNT6, is selected for study as its significant elevated-expression in breast cancer.
  • GALNT6 polypeptide N-acetylgalactosaminyltransferase 6
  • siRNA effectively inhibited expression of B7330N and significantly suppressed cell/tumor growth of breast cancer.
  • B7330N designed to UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 6, GALNT6, is selected for study as its significant elevated-expression in breast cancer.
  • GALNT6 polypeptide N-acetylgalactosaminyltransferase 6
  • siRNA effectively inhibited expression of B7330N and significantly suppressed cell/tumor growth of breast cancer.
  • novel protein B7330N promotes cell growth whereas cell growth is suppressed by small interfering RNAs corresponding to the B7330N gene.
  • small interfering RNAs corresponding to the B7330N gene show that B7330N protein stimulates oncogenic activity.
  • each of these novel oncoproteins is a useful target for the development of anti-cancer pharmaceuticals.
  • agents that block the expression of B7330N, or prevent its activity find therapeutic utility as anti-cancer agents, particularly anti-cancer agents for the treatment of breast cancers.
  • agents include antisense oligonucleotides, small interfering RNAs, and ribozymes against the B7330N gene, and antibodies that recognize B7330N.

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ES2364078T3 (es) 2011-08-24
WO2007013670A2 (en) 2007-02-01
CN101278060B (zh) 2011-09-07
JP5109063B2 (ja) 2012-12-26
US20090202543A1 (en) 2009-08-13
JP2009502111A (ja) 2009-01-29
US8795976B2 (en) 2014-08-05
DE602006021315D1 (de) 2011-05-26
CN101278060A (zh) 2008-10-01
EP1910570A2 (en) 2008-04-16

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